Analysis plots for FRC paper

Code by Friedrich Preusser (github.com/Fritze/)

All organoids acquired with Z1 lightsheet (August 2019).

Data shown here is all from Draq5 channel.

—

1. Multiview recontruction based on Draq5 signal.

2. 400x400 bounding box through full z.

3. Fuse (no downsampling, keeping anisotropy) and save.

4. Create new .hdf5 based on this small image and open in new project

5. FRC (no downsampling) with relative FRC, FFT size 200, stepsize=1 and not keeping anisotropy (FFT size 400 for confocal data)

- this will create 4 sub-tiles where FRC is calculated independently

- to assess FRC quality we always take the median of these 4 subtiles

6. For both Img and FRC measurements: Measurement per slice.

Select a tab to view it’s content

Code

list_data_csvs <- list.files(here("data"),".+csv$",include.dirs= FALSE)
offset_rollingmedian <- 51

read_data <- function(files){
  read.csv(files) %>%
  rename_all(tolower) %>%
  #extract protocol from beginning of file name
  mutate(protocol = gsub(".+\\_(.+)\\_org.+","\\1",basename(files))) %>%
  #extract measurement type
  mutate(measured = gsub("(^[^_]+).+","\\1",basename(files))) %>% 
  #extract OrganoidID (starting with "org" or "orgconf (for confocal data))
  mutate(orgID = gsub(".+\\_(org\\d+|orgconf\\d+).+","\\1",basename(files))) %>%
  #extract filename
  mutate(filename=basename(files)) %>%
  #extract angle
  mutate(angle=gsub(".+angle(\\d+).+","\\1",filename)) %>%
  mutate(angle=ifelse(angle == 0, "right",angle)) %>%
  mutate(angle=ifelse(angle == 1,"left",angle)) %>%
  #if angle is a digit number keep it otherwise make it NA
  mutate(angle = ifelse(grepl("angle",filename),angle,"both_angles")) %>%
  #all values (either quality or image feature measurements) should be named "value"
  rename_all(~sub('quality', 'value', .x)) %>%
  rename_all(~sub('mean','value',.x)) %>%
  #slice numbering in "slice" column
  mutate(slice = (1:n())-1)

}




data_full <- map_dfr(file.path(here("data"),list_data_csvs),read_data) %>% 
  rename(min_Img = min, max_Img = max) %>%
  select(protocol,orgID,slice,angle,measured, value,max_Img,min_Img) %>%
  mutate(measured = ifelse(measured == "Img-features","mean-intensity",measured)) %>%
  group_by(protocol,orgID,angle) %>%
  mutate(max_slice = max(slice)) %>%
  mutate(slice_norm = slice-max_slice/2) %>%
  group_by(protocol,orgID,angle,measured) %>%
  #contrast is just max minus minimum intensity at a given slice
  mutate(contrast = max_Img - min_Img) %>%
  mutate(contrast_norm = contrast / max(contrast)) %>%
  select(-c(max_Img,min_Img)) %>%
  #normalize all measured values between 0 and 1
  mutate(value_norm = value / max(value)) %>% 
  group_by(slice,orgID,protocol,angle) %>%
  ungroup() %>%
  arrange(protocol,orgID)



stepsizes_and_corresponding_orgIDs <- data.frame(
                    protocol = c("ClearT2","ClearT2","ClearT2",
                                  "Fructose","Fructose","Fructose",
                                  "ScaleA2","ScaleA2","ScaleA2",
                                 "Fructose","Fructose","Fructose",
                                 "Control","Control","Control"),
                    orgID = c("org1","org2","org3",
                              "org2","org4","org5",
                              "org1","org2","org3",
                              "orgconf1","orgconf2","orgconf3",
                              "orgconf1","orgconf2","orgconf3"),
                    stepsize = c(2.42, 2.42, 2.42,
                                1.7,1.45,1.55,
                                2.42,2.42,2.42,
                                2,2,2,
                                2,2,2))

data_full <- data_full %>%
  left_join(stepsizes_and_corresponding_orgIDs,by=c("protocol","orgID")) %>%
  mutate(slice_norm_micro = slice_norm * stepsize)

Plot for Figure 2

#select organoid to plot
selected_organoid <- "ScaleA2_org2"
boundaries <- c(15,70,210,260)

selected_slices <- c(7,60,134,220)


boundaries_micron <- filter(data_full,slice %in% boundaries) %>%
  #make name
  mutate(name = paste0(protocol,"_",orgID)) %>%
  #only the organoid we want to plot
  filter(name == selected_organoid) %>%
  distinct(slice_norm_micro) %>%
  pull(slice_norm_micro)

selected_slices_micron <- filter(data_full,slice %in% selected_slices) %>%
  #make name
  mutate(name = paste0(protocol,"_",orgID)) %>%
  #only the organoid we want to plot
  filter(name == selected_organoid) %>%
  distinct(slice_norm_micro) %>%
  pull(slice_norm_micro)

#dataframe describing edge locations
rects <- data.frame(xmin = c(boundaries_micron[1],boundaries_micron[2],boundaries_micron[3]),
                    xmax = c(boundaries_micron[2],boundaries_micron[3],boundaries_micron[4]),
                    ymin = -Inf,  
                    ymax = Inf,
                    fill = c( wes_palette("Moonrise3")[3], wes_palette("Moonrise3")[1], wes_palette("Moonrise3")[3]))

#filter data for plots
data_to_plot <- data_full %>%
  #only multiview reconstructed images
  filter(angle == "both_angles") %>%
  #make name
  mutate(name = paste0(protocol,"_",orgID)) %>%
  #only the organoid we want to plot
  filter(name == selected_organoid) 

intensity_plot <- data_to_plot %>%
  filter(measured == "mean-intensity") %>% 
  ggplot(., aes_string(x="slice_norm_micro",y="value"))+
  # geom_rect(data = rects, aes(xmin = xmin, xmax = xmax, ymin = ymin, ymax = ymax, fill = fill), inherit.aes = FALSE, alpha = 0.15) +
  geom_line(aes(color=name),size=1.5,color="black")  +
  geom_vline(xintercept=rects$xmin, size=0.3,alpha = 0.15) +
  geom_vline(xintercept=rects$xmax, size=0.3,alpha = 0.15) +
  theme_bw() +
  labs(y = "pixel\nintensity")+
  ggtitle("intensity") +
  scale_x_continuous(limits = c(min(data_to_plot$slice_norm_micro),max(data_to_plot$slice_norm_micro)),
                     breaks = c(-300,-200,-100,0,100,200,300))+
  theme(axis.title.x=element_blank(),
        axis.text.x=element_blank(),
        plot.title = element_text(size=10))+
  geom_vline(xintercept=selected_slices_micron,size=0.15,color="black")


contrast_plot <- data_to_plot %>%
  filter(measured == "mean-intensity") %>% 
  ggplot(., aes_string(x="slice_norm_micro",y="contrast"))+
  # geom_rect(data = rects, aes(xmin = xmin, xmax = xmax, ymin = ymin, ymax = ymax, fill = fill), inherit.aes = FALSE, alpha = 0.15) +
  geom_line(aes(color=name),size=1.5,color="black")  +
  geom_vline(xintercept=rects$xmin, size=0.3,alpha = 0.15) +
  geom_vline(xintercept=rects$xmax, size=0.3,alpha = 0.15) +
  theme_bw() +
  labs(y="contrast")+
  ggtitle("Contrast (raw)") +
  scale_x_continuous(limits= c(min(data_to_plot$slice_norm_micro),max(data_to_plot$slice_norm_micro)),
                     breaks = c(-300,-200,-100,0,100,200,300))+
  theme(axis.title.x=element_blank(),
        axis.text.x=element_blank(),
        plot.title = element_text(size=10))+
  geom_vline(xintercept=selected_slices_micron,size=0.15,color="black")


DCT_Shannon_entropy_plot <- data_to_plot %>%
  filter(measured == "DCT-Shannon-entropy") %>%
  ggplot(., aes_string(x="slice_norm_micro",y="value"))+
  #geom_rect(data = rects, aes(xmin = xmin, xmax = xmax, ymin = ymin, ymax = ymax, fill = fill), inherit.aes = FALSE, alpha = 0.15) +
  geom_line(aes(color=name),size=1.5,color="black")  +
  geom_vline(xintercept=rects$xmin, size=0.3,alpha = 0.15) +
  geom_vline(xintercept=rects$xmax, size=0.3,alpha = 0.15) +
  theme_bw() +
  labs(y="DCT shannon entropy",x="µm (relative to center)") +
  ggtitle("DCT shannon entropy") +
  scale_x_continuous(limits= c(min(data_to_plot$slice_norm_micro),max(data_to_plot$slice_norm_micro)),
                     breaks = c(-300,-200,-100,0,100,200,300))+
  theme(axis.title.x=element_blank(),
        axis.text.x=element_blank(),
        plot.title = element_text(size=10))+
  geom_vline(xintercept=selected_slices_micron,size=0.15,color="black")
 


FRC_plot_relative <- data_to_plot %>%
  filter(measured == "FRC") %>%
  ggplot(., aes_string(x="slice_norm_micro",y="value"))+
  #geom_rect(data = rects, aes(xmin = xmin, xmax = xmax, ymin = ymin, ymax = ymax, fill = fill), inherit.aes = FALSE, alpha = 0.15) +
  geom_line(aes(color=name),size=1.5,color="black")  +
  geom_vline(xintercept=rects$xmin, size=0.3,alpha = 0.15) +
  geom_vline(xintercept=rects$xmax, size=0.3,alpha = 0.15) +
  theme_bw() +
  ggtitle("Relative FRC") +
  scale_x_continuous(limits= c(min(data_to_plot$slice_norm_micro),max(data_to_plot$slice_norm_micro)),
                     breaks = c(-300,-200,-100,0,100,200,300))+
  theme(plot.title = element_text(size=10))+
  geom_vline(xintercept=selected_slices_micron,size=0.15,color="black")





#put all plots together
patch <- (intensity_plot  / contrast_plot  / DCT_Shannon_entropy_plot / FRC_plot_relative )

#plot
plot <- patch & theme(panel.border = element_rect(size=2),
              axis.text = element_text(face="bold",size=12),
              panel.background = element_rect(fill = "transparent"), # bg of the panel
              plot.background = element_rect(fill = "transparent", color = NA), # bg of the plot
              panel.grid.major = element_blank(), # get rid of major grid
              panel.grid.minor = element_blank(), # get rid of minor grid
              )


ggsave(plot=plot, file="Figure_2_lineplots.png", type = "cairo-png",  bg = "transparent",dpi = 300,width=12)

## Saving 12 x 5 in image

plot

Figure 3 - lightsheet data

line_plot <- function(data_to_plot,X,Y,xlab,ylab,colorby){
  ggplot(data_to_plot, aes_string(x=X,y=Y,group="name"))+
    geom_line(aes_string(color=colorby),size=1) + 
    labs(x=xlab, y=ylab) +
    facet_grid(~protocol,scale="free_x")+
    theme_bw() +
    theme(legend.position = "top")
}

unfused_examples <- data_full %>%
  mutate(name = paste0(protocol,"_",orgID)) %>%
  filter(!grepl("both_angles",angle)) %>% 
  distinct(name) %>%
  pull(name)
  
selected_slices_micron <- c(0,200)

data_to_plot <- data_full %>%
  mutate(name = paste0(protocol,"_",orgID)) %>%
  filter(name %in% unfused_examples) %>%
  filter(!grepl("ScaleA2",name)) %>% 
  #select specific organoids
  filter(name %in% c("ClearT2_org2", "Fructose_org4")) %>% 
  mutate(name = paste0(protocol,"_",orgID)) %>%
  mutate(microns_from_start = slice * stepsize) %>%
  mutate(angle = ifelse(!grepl("both_angles",angle),angle,"multiview fusion"))

FRC_plot <- data_to_plot %>%
  filter(measured == "FRC") %>% 
  #filter out slices with value == 0
  filter(value > 0) %>% 
  line_plot(.,"slice_norm_micro","value","µm (relative to center)","FRC score (raw)","name") +
  facet_grid(~angle,scale="free_x")


#put everything together
patch <-  FRC_plot


#plot
plot <- patch & 
    theme(panel.border = element_rect(size=1.5),
              axis.text = element_text(face="bold",size=12),
              strip.text = element_text(face="bold",size=14)
              ) &
    scale_color_manual(values = wes_palette("Moonrise3")) &
    geom_vline(xintercept=selected_slices_micron,linetype='dashed',size=0.8,color="red")

ggsave(plot=plot, file="Figure_3_lightsheet.png", type = "cairo-png",  bg = "transparent",dpi = 300,height=4,width=8)

plot

Figure 4 - confocal data

replicates_and_corresponding_orgIDs <- data.frame(protocol =
                                          c("Fructose","Fructose","Fructose",
                                            "Control","Control","Control"),
                    orgID = c("orgconf1","orgconf2","orgconf3",
                              "orgconf1","orgconf2","orgconf3"),
                    replicates = c("replicate 3", "replicate 2", "replicate 1",
                                  "replicate 3","replicate 2","replicate 1"))

selected_slices_micron <- c(150)


data_to_plot <- data_full %>%
  filter(grepl("conf",orgID)) %>%
  left_join(replicates_and_corresponding_orgIDs,by=c("orgID","protocol")) %>%
  mutate(protocol = ifelse(protocol == "Control", "Uncleared",protocol)) %>%
  mutate(name = paste0(protocol,"_",orgID)) %>%
  mutate(microns_from_start = slice * stepsize)

FRC_plot <- data_to_plot %>%
  filter(measured == "FRC") %>% 
  line_plot(.,"microns_from_start","value_norm","µm inside","FRC score (normalized)","replicates")+
    geom_hline(yintercept=0.5,linetype='dashed',size=0.8,color="black",alpha=0.7)



#put everything together
patch <- FRC_plot


#plot
plot <- patch & 
    theme(panel.border = element_rect(size=1.5),
              axis.text = element_text(face="bold",size=12),
              strip.text = element_text(face="bold",size=14)
              ) &
    scale_color_manual(values = wes_palette("Moonrise3"))

ggsave(plot=plot, file="Figure_3_confocal.png", type = "cairo-png",  bg = "transparent",dpi = 300,height=4,width=8)

plot

Line Plots for Figure 4

selected_slices_micron <- c(-200,0)


replicates_and_corresponding_orgIDs <- data.frame(protocol = c("ClearT2","ClearT2","ClearT2",
                                  "Fructose","Fructose","Fructose",
                                  "ScaleA2","ScaleA2","ScaleA2"),
                    orgID = c("org3","org2","org1",
                              "org5","org4","org2",
                              "org2","org3","org1"),
                    replicates = c("replicate 1", "replicate 2", "replicate 3",
                                  "replicate 1","replicate 2","replicate 3",
                                  "replicate 1","replicate 2","replicate 3"))

  

data_to_plot <- data_full %>%
  left_join(replicates_and_corresponding_orgIDs,by=c("orgID","protocol")) %>%
  filter(angle == "both_angles") %>%
  mutate(name = paste0(protocol,"_",orgID)) %>%
  #filter out confocal data
  filter(!grepl("orgconf",orgID))


contrast_plot <- data_to_plot %>%
  filter(measured == "mean-intensity") %>%
  line_plot(.,"slice_norm_micro","contrast","slice","Contrast","replicates")+
    labs(y="Contrast",x="µm (relative to center)")


DCT_Shannon_entropy_plot <- data_to_plot %>%
  filter(measured == "DCT-Shannon-entropy") %>%
  line_plot(.,"slice_norm_micro","value","slice","DCT Shannon entropy","replicates")+
    labs(y="Entropy",x="µm (relative to center)")

FRC_plot <- data_to_plot %>%
  filter(measured == "FRC") %>% 
  line_plot(.,"slice_norm_micro","value","slice","FRC score (raw)","replicates") +
  labs(y="FRC",x="µm (relative to center)")


#put all plots together
patch <- contrast_plot / DCT_Shannon_entropy_plot / FRC_plot 


#plot
plot <- patch & 
    theme(panel.border = element_rect(size=1.5),
              axis.text = element_text(face="bold",size=12),
              strip.text = element_text(face="bold",size=14)
              ) &
    scale_x_continuous(limits= c(min(data_to_plot$slice_norm_micro),max(data_to_plot$slice_norm_micro)),
                     breaks = c(-300,-150,0,150,300)) &   
    scale_color_manual(values = wes_palette("Moonrise3")) &
    geom_vline(xintercept=selected_slices_micron,linetype='dashed',size=0.8,color="red")

ggsave(plot=plot, file="Figure_4_lineplots.png", type = "cairo-png",  bg = "transparent",dpi = 300,height=8,width=16)

plot

Box Plot for Figure 4

#make sampling reproducible
set.seed(2020)

#take data_to_plot dataframe from previous chunk
#and select 200 slices per experiment (x 3 replicates x 3 protocols = 1800 slices in total)
selected_slices <- data_to_plot %>%
  mutate(name_slice = paste0(name,"_",slice)) %>%
  group_by(orgID,protocol) %>%
  sample_n(200) %>%
  pull(name_slice)

#filter out FRC for only those slices
data_to_plot2_FRC <- data_to_plot %>%
  mutate(name_slice = paste0(name,"_",slice)) %>%
  filter(measured == "FRC") %>%
  filter(name_slice %in% selected_slices)

boxplot_FRC <- ggplot(data_to_plot2_FRC, aes(y=value,x=protocol,group=protocol,fill=protocol))+
  geom_violin(fill="lightgrey",alpha=0.8,size=0,inherit_aes=FALSE,outlier.shape=NA)+
  geom_boxplot(width=0.25,size=1,alpha=.3,outlier.alpha = 1) +
  scale_y_continuous(limits=c(0,max(data_to_plot2_FRC$value + 20)))+
  geom_signif(comparisons = list(c("Fructose", "ScaleA2"),c("ClearT2","Fructose")), 
              map_signif_level=TRUE,y_position = c(74, 81),test = "wilcox.test",size=1)+
  geom_signif(comparisons = list(c("ClearT2","ScaleA2")),
              map_signif_level = TRUE,y_position=c(88),test = "wilcox.test",size=1)+
  labs(x="protocol", y="FRC") +
  scale_colour_discrete(name = "Organoid ID") +
  theme_bw() +
  theme(legend.position = "right")+
  scale_fill_brewer(palette = "Set2")+
  coord_flip()

#filter out DCT Shannon entropy values for the same slices
#so that we look at the same images in both plots
data_to_plot2_DCT_Shannon_entropy <- data_to_plot %>%
  mutate(name_slice = paste0(name,"_",slice)) %>%
  filter(measured == "DCT-Shannon-entropy") %>%
  filter(name_slice %in% selected_slices)

boxplot_DCT_Shannon_Entropy <- ggplot(data_to_plot2_DCT_Shannon_entropy, aes(y=value,x=protocol,group=protocol,fill=protocol))+
  geom_violin(fill="lightgrey",alpha=0.8,size=0,inherit_aes=FALSE,outlier.shape=NA)+
  geom_boxplot(width=0.25,size=1,alpha=.3,outlier.alpha = 1) +
  scale_y_continuous(limits=c(0,max(data_to_plot2_DCT_Shannon_entropy$value + 0.007)))+
  geom_signif(comparisons = list(c("Fructose", "ScaleA2"),c("ClearT2","Fructose")),
              map_signif_level=TRUE,y_position = c(0.015, 0.02),test = "wilcox.test",size=1)+
  geom_signif(comparisons = list(c("ClearT2","ScaleA2")),
  map_signif_level = TRUE,y_position=c(0.023),test = "wilcox.test",size=1)+
  labs(x="protocol", y="Entropy") +
  scale_colour_discrete(name = "Organoid ID") +
  theme_bw() +
  theme(legend.position = "right")+
  scale_fill_brewer(palette = "Set2")+
  coord_flip()


plot <- boxplot_FRC & theme(panel.border = element_rect(size=1.5),
              axis.text = element_text(face="bold",size=12),
              legend.direction  = "vertical",
              legend.key.height =unit(2, "cm"),
              legend.key.width = unit(1,"cm"))

ggsave(plot=plot, file="Figure_4_FRC_boxplot.png", type = "cairo-png",  bg = "transparent",dpi = 300,height=3.5,width=7)

plot

plot <- boxplot_DCT_Shannon_Entropy & theme(panel.border = element_rect(size=1.5),
              axis.text = element_text(face="bold",size=12),
              legend.direction  = "vertical",
              legend.key.height =unit(2, "cm"),
              legend.key.width = unit(1,"cm"))

ggsave(plot=plot, file="Figure_4_DCT-Shannon-Entropy_boxplot.png", type = "cairo-png",  bg = "transparent",dpi = 300,height=3.5,width=7)

plot